Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
  • C12N5/0629—Keratinocytes; Whole skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
  • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
  • A61F2/02—Prostheses implantable into the body
  • A61F2/10—Hair or skin implants
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
  • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
  • A61L27/60—Materials for use in artificial skin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

• the present invention relates to the growth of human epidermal cells to produce useful sheet-like products, the resultant products, and use thereof, for example, as skin graft materials.
• the present invention relate in part to the dissociation of epidermis into epidermal cells and the growth of the epidermal cells into a layer of epidermis consisting essentially of epidermal cells which do not express immune stimulatory antigens.
• Another aspect of the present invention involves sheet-like products comprising at least one layer of epidermis grown in culture and consisting essentially of non-immune-competent epidermal cells and the use thereof as a skin grafting material.
• Eisinger et al Human Epidermal Cell Cultures: Growth and Differentiation in the Absence of Dermal Components or Medium Supplements
• Proc. Natl. Acad. Sci. USA volume 76, No. 10, pp. 5340-5344 (October, 1979).
• Eisinger et al "Wound Coverage by a Sheet of Epidermal Cells Grown In Vitro from Dispersed Single Cell Preparations," Surgery, St. Louis, Volume 88, No. 2, pp. 287-293 (March, 1980).
• the above described prior art process of Eisinger and Hefton may be summarized as broadly involving the sequence of separating the epidermis in human skin from the dermis, dissociating the epidermis into epidermal cells and growing the epidermal cells in a tissue culture medium.
• Specific process conditions are disclosed by Eisinger et al for use in each of the above steps and certain of these process conditions will be discussed in greater detail hereinbelow in relationship to a corresponding proces step in the cell growth process embodiments of the present invention.
• ATPase staining was used as an analytical tool to determine the presence of immune-competent Langerhans cells, which are included in the cells carrying surface antigens which cause transplantation rejection.
• the products of Eisinger and Hefton contain 2 to 3% ATPase positive cells.
• Morhenn et al "Cultured Human Epidermal Cells Do Not Synthesize HLA-Dr", The Journal of Investigative Dermatology, 78:32-37 (1982) state that they obtained epidermal cell cultures grown on collagen gel or gelatin membranes free of the immune stimulatory antigen (HLA-Dr) and also free of Langerhans cells, following a culturing period of 7 days or longer. It is the present inventor's belief that the absence of HLA-Dr immunogens in Morhenn's product is due to a combination of culturing parameters, probably including the use of the collagen or gelatin substrate and not due solely to the duration of culturing.
• Morhenn et al (1) state that their cultured epidermal cells include some ATPase positive cells, which would be expected to be Langerhans cells, but if not could be a type of non-Langerhans dendritic cell possessing some immunocompetence, and (2) do not describe a process for producing an integral self-supporting sheet consisting of epidermal cells.
• the composite structure of Morhenn, including the collagen or gelatin substrate, would in itself be poorly suitable as a skin graft.
• an object of the present invention to provide an epidermal sheet formed of confluent or associated human epidermal cells, useful as a skin graft.
• Another object of the present invention is to provide an integral, self-supporting epidermal sheet formed of essentially one or more layers of ATPase negative human epidermal cells.
• Still another object of the present invention is to provide an integral, self-supporting epidermal sheet formed essentially of one or more layers of ATPase negative human epidermal cells, which cells do not exhibit HLA-Dr immune stimulatory antigens.
• a further object of the present invention is to provide an epidermal sheet usable as an allograft or homograft, that is for non-donor-related patients.
• Another object of the present invention is to provide processes for culturing human epidermal cells to produce integral, self-supporting sheets of one or more layers of human epidermal cells, which sheets are essentially free of both ATPase positive cells and other cells which contain transplantation immune competent antigens.
• Still another object of the present invention is to provide processes for treating burn victims or other patients needing skin grafts in which an epidermal sheet as disclosed herein is applied to affected areas.
• Other uses for the epidermal sheet of the present invention will be apparent to the skilled artisan, some of which will be discussed hereinbelow.
• a process for growing human epidermal cells in vitro in the absence of dermal components to form an integral confluent, self-supporting sheet of one or more layers of pure human epidermis free of immune-competent cells wherein dissociated epidermal cells are treated in a manner enabling selective removal of essentially all of the immune-competent cells prior to tissue growth.
• the cells are treated enzymatically to disrupt essentially all immune-competent cells, including the cells exhibiting HLA-Dr antigens and including all ATPase-positive cells.
• the dissociated epidermal cells are treated with a protein material which selectively attaches to the immune-competent cells, including the ATPase-positive cells.
• a single cell suspension of epidermal cells containing ATPase-positive cells and/or other immune stimulatory cells is incubated in the presence of deoxyribonuclease (DNase), the intact cells are collected and then the collected cells are cultured to yield a self-supporting sheet of pure epidermal tissue.
• DNase deoxyribonuclease
• a single cell suspension of epidermal cells containing ATPase-positive cells and/or other immune stimulatory cells is incubated in the presence of tobacco glycoprotein (TGP), the epidermal cells to which the TGP does not attach are collected, and the collected cells are cultured to yield a self-supporting sheet of pure epidermal tissue.
• TGP tobacco glycoprotein
• the collected cells are grown in a plastic flask in a tissue culture medium having a pH of about 6.5 to 7.2.
• human skin is obtained from a suitable donor such as cadavers, unrelated to possible skin graft recipients. Therefore, the skin graft products of this invention can be produced in quantity, packaged and stored under conditions providing availability upon need without concern as to immune-rejection by the recipient.
• epidermis of human skin can be separated from the dermis using prior art techniques.
• concentration of trypsin is elevated beyond the levels believed to be taught in the prior art.
• the aforementioned Eisinger and Hefton procedure and Morhenn et al article disclose the use of 0.25% and 0.3% trypsin solutions to facilitate separation of dermis from epidermis.
• the present inventor's preferred procedure employs incubation in a 0.5% trypsin solution.
• the recommended step is to incubate at about 32 to 40° C., preferably about 37° C., for about 45 to 120 minutes, preferably about 90 minutes, in about a 0.3 to 0.75% trypsin solution, preferably about 0.5% trypsin.
• the epidermis is dissociated into essentially single cells to form a single cell suspension of epidermal cells in a liquid medium. Concommitantly with dissociation, or thereafter but prior to significant tissue sheet growth, the epidermal cells are treated to enable selective removal of the immune-competent cells from the overall epidermal cell population.
• the present invention comprises two process techniques to enable removal of the ATPase-positive and other immune-competent cells from remaining epidermal cells.
• these two broad embodiments are disclosed herein for practice one to the exclusion of the other, it is believed that without undue experimentation, a combined simultaneous or sequential process utilizing both techniques could be operable.
• the preferred process embodiment at this time to enable separation of the undesirable ATPase-positive and other immune-competent cells from the remainder involves the incubation of the epidermal single cells in DNase.
• the single cells can be conveniently dispersed in an aqueous solution of about 0.1 to 0.6% DNase, and held therein at a temperature of about 18 to 37° C., preferably about 20 to 25° C. for about 10 to 60 minutes, with vigorous agitation such as by stirring with a sterile glass rod.
• the above suggested DNase concentrations are based on use of D 0751 (DN-100) DNase available from Sigma (St. Louis, Mo.), having an activity of approximately 1,4000 Kunitz units per milligram protein.
• Adjustment in DNase concentration should be made based on activity of the DNase employed, through routine calculation and experimentation.
• Conventional analytical techniques such as immunofluorescent staining for HLA-Dr antigens, ultrastructural identification of Langerhans cells, ATPase staining and the like can be employed to monitor the DNase incubation step. It is expected that the concentration of DNase toward the lower range above defined would involve longer incubation times than the employment of higher DNase concentrations.
• the recommended DNase concentration is approximately 0.5%.
• the DNase can be dissolved in an isotonic medium, such as MEM, PBS, normal saline, etc. The present inventor has found that the use of a 0.025% DNase incubation for about 30 minutes at about 21° C.
• HLA-Dr antigen which cells should stimulate allogenic T cells, after two days of culturing, as compared to 5 to 7% of said cells prior to culturing. It is noted that the ability of epidermal cells to stimulate the proliferation of allogenic T-lymphocytes in a MSLR (mixed skin cell lymphocyte reaction) can serve as one measure of histoincompatibility.
• the second technique for removal of the undesirable ATPase positive and other immune stimulatory cells involves contacting the epidermal single cells with a glycoprotein which selectively attaches to the undesirable immune-competent cells. Simultaneously with or following attachment, the cells having the glycoprotein attached thereto are separated from the desired (non immune stimulatory) cells, which cells do not associate with the glycoprotein.
• the preferred glycoprotein is tobacco glycoprotein (TGP), which is described in detail by Becker et al, "Tobacco, Cocoa, Coffee, and Ragweed: Cross-Reacting Allergens That Activate Factor-XII-Dependent Pathways," Blood, Volume 58, No. 5, p. 861 (1981).
• TGP protein materials
• rutin rutin, cocoa antigen, etc.
• a suggested process which has been employed involving the use of TGP is to attach the TGP to a solid phase, such as glass beads or a Sepharose column, bring the solid phase into contact with the single cell suspension, such as by passing the suspension through a TGP-Sepharose column, and recovering the cells remaining dissociated from the solid phase (affinity chromatography).
• filtration can be carried out using a filtering medium allowing passage of whole cells, while retaining clumps of debris larger than the single cells.
• a sterile gauze filter can be employed. Thereafter, the cells can be collected and concentrated by centrifugation, for example for 10 minutes at 200 g.
• tissue culture medium Following the removal of the undesirable ATPase-positive and other immune-competent cells, the remaining epidermal cells are grown in a tissue culture medium.
• tissue growth mediums and processes such as those of Eisinger and Hefton, disclosed above, can be employed at this stage, it has been found that tissue growth increases where the pH of the culture medium is controlled to be about pH 6.5 to 7.2.
• the recommended tissue culture growth conditions at this time are to utilize a temperature of around 37° C., atmosphere of 100% humidity, a cell culture medium containing about 20% fetal calf serum, with the cells being seeded into plastic tissue culture vessels to a final density of about 10 5 to 10 8 cells per ml of culture medium.
• One milliliter of culture medium is added to each flask per 5 cm 2 of surface area of the flask covered by the culture medium.
• the seeding density can be expressed as 10 5 to 10 8 cells per 5 cm 2 of flask surface area.
• Subculturing can be carried out for about 7 to 21 days, depending upon the thickness of tissue sheet desired.
• an epidermal sheet consisting of a single or monolayer of epidermal cells could prove useful, while in most cases involving the formation of grafting products, a multi-layered integral epidermal sheet would be preferred.
• a single layer product or a product having several layers of cells will be used depending on wound depth, wound size and the like factors.
• the epidermal sheet attaches to the plastic culture flask.
• an enzyme which preferentially acts at the tissue sheet-plastic interface can be employed, for example dispase in a concentration of about 0.1 to 1.0%.
• the dispase concentration will depend on the specific activity of the enzyme, the dispase used herein having a specific activity of 0.5 units per milligram lyophilized enzyme. Routine experimentation would indicate the correct amount of the enzyme to be used.
• a transfer member such as the dermal side of pig skin, a collagen sponge, a sheet of polyvinylidene polymer, polyester or hydrogel, or Vaseline-impregnated gauze, can be used to transfer the epidermal sheet from the plastic growth flask.
• the epidermal sheet is floated to such a transfer member for removal or floated directly to the area of application, to, for example, an afflicted area on a burn patient.
• a notable advantage of the present invention is the provision of mono- or multi-layered epidermal sheets consisting of human epidermal cells which do not contain immune stimulatory antigens.
• the term "essentially free of immune-competent cells” means that the level of immune-competent cells is so low that immunorejection does not occur when the tissue is used as a skin graft for non-related recipients.
• the term "essentially free of ATPase-positive cells” means that the level of ATPase positive cells is below that causing rejection when the tissue is used as a skin graft for an unrelated recipient.
• the Examples presented hereinbelow provided epidermal cell tissues which did not contain any immunecompetent cells as tested by immunofluorescent staining and by ATPase staining. Thus, transplantation can be carried out for burn patients who are unrelated to the donor, which can be a cadaver.
• epidermal sheets provided by the present invention, such as for drug testing and the like, i.e., testing of various materials such as shampoos and cosmetics which contact the skin.
• Partial-thickness pieces of skin were removed with a keratome and immersed in Eagle's minimum essential medium at 4° C. (MEM) (Grand Island Biological Co., Grand Island, N.Y.) with 1,000 U penicillin/ml, 1,000 mcg streptomycin/ml, 25 mcg Fungizone/ml and 10% Fetal Bovine Serum (FBS).
• MEM Eagle's minimum essential medium
• FBS Fetal Bovine Serum
• the epidermal layers were mechanically separated from the underlying dermis and dispersed to single cells in 0.5% DNase (DO751 (DN-100), Sigma, St. Louis, Mo.) in PBS and held in the DNase with vigorous agitation at room temperature for about 30 minutes.
• DNase DO751 (DN-100), Sigma, St. Louis, Mo.
• CM complete medium
• FBS complete medium
• 100 U penicillin/ml 100 mcg streptomycin/ml
• 2.5 mcg Fungizone/ml 0.1 mM hydrocortisone (Merck, Sharp and Dohme, West Point, Pa.)
• the viability of cells prepared in this manner was 90 to 95% by trypan blue dye exclusion.
• Cells were seeded into plastic tissue culture flasks at a final density of 2 ⁇ 10 5 to 10 8 cells per ml of culture medium.
• the cultures were incubated at 37° C. in 100% humidity in a 95% air/5% CO 2 environment.
• the medium was changed every three days and the cultures became confluent after 14 to 21 days.
• the cultured epidermal cells may be removed from the tissue culture flask any time after confluency, for example after 3 to 4 weeks in this particular instance. Prior to the removal from the flasks, the cells were incubated in MEM without FBS for 24 hours.
• Partial-thickness pieces of skin were removed with a keratome and immersed in Eagle's minimum essential medium at 4° C. (MEM) (Grand Island Biological Co., Grand Island, N.Y.) with 1,000 U penicillin/ml, 25 mcg Fungizone/ml and 10% Fetal Bovine Serum (FBS).
• MEM Eagle's minimum essential medium at 4° C.
• FBS Fetal Bovine Serum
• TGP Tobacco glycoprotein
• rutin-BSA rutin conjugated with bovine serum albumin
• C MEM plus 2 mM 1-glutamine (GIBCO), 20% FBS, 100 U penicillin/ml, 100 mcg streptomycin/ml, 2.5 mcg Fungizone/ml and 0.1 mM hydrocortisone (Merck, Sharp and Dohme, West Point, Pa.) at pH 5.8 to 6.0.
• C MEM plus 2 mM 1-glutamine (GIBCO), 20% FBS, 100 U penicillin/ml, 100 mcg streptomycin/ml, 2.5 mcg Fungizone/ml and 0.1 mM hydrocortisone (Merck, Sharp and Dohme, West Point, Pa.) at pH 5.8 to 6.0.
• the viability of cells prepared in this manner was 90 to 95% by trypan blue dye exclusion.
• Cells were seeded into plastic tissue culture flasks at a final density of 10 5 to 10 8 cells per ml of culture medium. The cultures were incubated at 37° C.
• the cultured epidermal cells may be removed from the tissue culture flask at any time after confluency with a neutral protease such as dispase.
• the absence of immune competent cells was determined by two testing procedures (the cell population can be tested as single cell suspensions prior to sheet growth), with the cell population of the present invention containing zero per cent immune competent cells per both testing procedures.
• cultured cells are fixed in a 6% solution of cold neutral formalin.
• the cells are then incubated in 5 ml of 1.25% sodium glycerophosphate in 0.2 M tris buffer mixed with 30 ml 0.2% lead nitrate and 5 ml distilled water for 180 minutes at 37° C.
• the cultured cells are washed in tap water and dipped in dilute ammonium sulfide for several minutes.
• the preparations are covered with glycerin prior to observation by light microscopy.
• the cells containing ATPase activity are revealed by black deposits of lead sulfide.
• Antibody binding was visualized by incubating the cells with commercially available fluonuescein isothiocyanate (FITC)-conjugated F(ab') 2 fragments from goat antiserum directed against rabbit IgG molecules for 30 minutes at 4° C. After this incubation period, the cells were washed twice with PBS-BSA and collected by centrifugation (200 g. 10 minutes). The cell pellets were resuspended in a small volume of PBS-BSA and then examined for fluorescence under a sealed cover slip with a Leitz Ortholux microscope equipped for incident illumination.
• FITC fluonuescein isothiocyanate
• the pure epidermal cell sheets of the present invention have been used for grafting to non-related recipients.
• a procedure utilized has been to excise and cover second degree burn wounds with sterile pigskin for 3 to 4 days to permit a bed of early granulation tissue to form.
• the pig skin is then removed and the cultured epidermal cell sheet placed on the granulation tissue with either side of the sheet facing the wound.
• the wound is then dressed with wet to wet moist dressings.
• the culture grafts may be redressed with occlusive or semi-occlusive dressings after the epidermal cells have adhered 24 to 48 hours after transplanation.
• the grafted area healed similar to second degree burn areas grafted with conventional split-thickness autografts, without any indication of rejection.

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• 1983
  • 1983-06-14 US US06/504,190 patent/US4769317A/en not_active Expired - Fee Related
• 1984
  • 1984-06-14 EP EP19840902429 patent/EP0148868A4/en not_active Withdrawn
  • 1984-06-14 AU AU30645/84A patent/AU3064584A/en not_active Abandoned
  • 1984-06-14 JP JP59502377A patent/JPS60501556A/ja active Pending
  • 1984-06-14 WO PCT/US1984/000916 patent/WO1985000042A1/en not_active Application Discontinuation

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